Project Summary Small RNA second messenger molecules are quickly becoming recognized as potent immune regulatory factors with therapeutic potential. In humans, the endogenous second messenger 2?3? cGAMP is generated by the enzyme cGAS (cyclic GMP?AMP synthase) in response to double stranded DNA sensing in the cytoplasm. The source of cGAS-activating DNA can be replicating intracellular pathogens or as a result of cellular stress, inappropriate separation of genomic DNA during cell division, or tumor cell DNA delivered by uptake of dying cell debris in cancer. STING (Stimulator of Interferon Genes) is an essential adaptor molecule in the human innate immune system capable of triggering downstream transcription of type-I interferon genes and proinflammatory cytokines as one of the earliest cellular responses to infection by pathogenic bacteria and viruses. STING downstream signaling responses are directly dependent on its ability to detect bacterial cyclic dinucleotides and human derived cGAMP? effectively integrating bacterial, viral, and autoimmunity pathways. cGAS is a recently identified and characterized member of a sub-family of human nucleotidyltransferase enzymes known as Mab-21. The surprisingly well conserved and important role of cGAS second messenger signaling in animals suggests that other orphan Mab-21 family enzymes must be fulfilling similar functional purposes and likely generate novel RNA products. MB21D2 is a putative RNA second messenger synthase that has low sequence conservation with cGAS yet has high predicted structural homology. Differences within the DNA binding site between cGAS and MB21D2 imply a different mode of activation. I hypothesize that MB21D2 is not only a structural but also a potential functional homolog of human cGAS which can synthesize a novel second messenger in response to bacterial insult triggering a downstream immune response. I aim to test the functionality of purified recombinant MB21D2 using radiolabeled nucleotide substrates and screen a panel of potential activating ligands based on previous structural and binding studies on related homologs from bacteria. I have preliminary evidence that MB21D2 is an active enzyme but have yet to identify the product or products that it generates. Ongoing experiments are designed to use a yeast protein expression system to detect novel nucleotide products in lysates using high resolution mass spectrometry analysis. I also plan to determine the structure of human MB21D2 using X-ray crystallographic methods which will inform our understanding behind the mechanism of its activation. My other major aim is to observe the signaling consequences of MB21D2 overexpression, knockout, and potential activation by small molecules in the context of living cells. I plan to monitor transcriptional profiles of different human and mouse cell lines to look for changes in mRNA expression and search for hallmark signatures of innate immune signaling responses. I also plan to test for MB21D2 activation of immune response dependent on the STING pathway.